Control of RNA degradation is a critical cell function, and gene expression is often regulated at the level of RNA stability. See, e.g., Sha, G. and Kamen R., Cell, 46:659-667 (1986). Nevertheless, relatively little is known about the biochemical pathways that mediate RNA degradation in mammalian or plant systems. For instance, most if not all of the ribonucleases responsible for mRNA turnover in mammalian or plant cells remain unidentified. This was reviewed in Brawerman, G., Cell, 57:9-10 (1989).
Presently, the 2-5A system is believed to be the only well-characterized RNA degradation pathway from higher animals including man. See FIG. 1. See also, e.g., Kerr, I. M. and Brown, R. E., Prod. Natl. Acad. Sci. USA, 75:256-260 (1978) and Cayley, P. J. et al.: Biophys Res. Commun., 108:1243-1250 (1982); reviewed in Sen, G. C. and Lengyel, P., J. Biol. Chem., 267:5017-5020 (1992). The activity of the 2-5A system is believed to be mediated by an endoribonuclease known as 2-5A-dependent RNase. See Clemens, M. J. and Williams, B. R. G., Cell, 13:565-572 (1978). 2-5A-dependent RNase is a unique enzyme in that it requires 2-5A, unusual oligoadenylates with 2',5' phosphodiester linkages, p.sub.n (A2'p).sub.n A, for ribonuclease activity. See Kerr, I. M. and Brown, R. E., Prod. Natl. Acad. Sci. USA, 75:256-260 (1978). 2-5A is produced from ATP by a family of synthetases in reactions requiring double-stranded RNA (dsRNA). See FIG. 1. See also Hovanessian, A. G. et al., Nature, 256:537-539 (1977); Marie, I. and Hovanessian, A. G., J. Bio. Chem., 267:9933-9939 (1992). 2-5A is unstable in cells and in cell-free systems due to the combined action of 2',5'-phosphodiesterase and 5'-phosphatase. See Williams, B. R. G. et al.; Eur. J. Biochem., 92:455-562 (1978); and Johnson, M. I. and Hearl, W. G., J. Biol. Chem., 262:8377-8382 (1987). The interaction of 2-5A-dependent RNase and 2-5A(K.sub.d =4.times.10.sup.-11 M), Silverman, R. H. et al., Biol. Chem., 263:7336-7341 (1988), is highly specific. See Knight, M. et al., Nature, 288:189-192 (1980). 2-5A-dependent RNase is believed to have no detectable RNase activity until it is converted to its active state by binding to 2-5A. See Silverman, R. H., Anal. Biochem., 144:450-460 (1985). Activated 2-5A-dependent RNase cleaves single-stranded regions of RNA 3' of UpNp, with preference for UU and UA sequences. See Wreschner, D. H. et al., Nature, 289:414-417 (1981a); and Floyd-Smith, G. et al., Science, 212:1020-1032 (1981). Analysis of inactive 2-5A-dependent RNase from mouse liver revealed it to be a single polypeptide of approximately 80 kDa. See Silverman, R. H. et al., Biol. Chem., 263:7336-7341 (1988).
Although full scope and biological significance of the 2-5A system remains unknown, studies on the molecular mechanisms of interferon action have provided at least some of the functions. Interferons .alpha., or Y are believed to induce the accumulation of both 2-5A-dependent RNase, Jacobsen, H. et al., Virology, 125:496-501 (1983A) and Floyd-Smith, G., J. Cellular Biochem., 38:12-21 (1988), and 2-5A synthetases, Hovanessian, A. G. et al., Nature, 268:537-539 (1977), reviewed in Sen, G. C. and Lengyel, P., J. Biol. Chem., 267:5017-5020 (1992). Furthermore, several investigations have implicated the 2-5A system in the mechanism by which interferon inhibits the replication of picornaviruses. Indeed, 2-5A per se and highly specific 2-5A mediated rRNA cleavage products were induced in interferon-treated, encephalomyocarditis virus (EMCV)-infected cells. See Williams, B. R. G., Nature, 282:582-586 (1979); Wreschner, D. H. et al., Nucleic Acids Res., 9:1571-1581 (1981b); and Silverman, R. H. et al., Eur. J. Biochem., 124:131-138 (1982a). In addition, expression of 2-5A synthetase cDNA inhibited the replication of picornaviruses, Chebath, J., Nature, 330:587-588 (1987) and Rysiecki, E. F. et al., J. Interferon Res., 9:649-657 (1989), and the introduction of a 2-5A analogue inhibitor of 2-5A-dependent RNase into cells reduced the interferon-mediated inhibition of EMCV replication. See Watling, D. et al., EMBO J., 4:431-436 (1985). Further, 2-5A-dependent RNase levels were correlated with the anti-EMCV activity of interferon, Kumar, R. et al., J. Virol., 62:3175-3181 (1988), and EMCV-derived dsRNA both bound to and activated 2-5A synthetase in interferon-treated, infected cells. See Bribaudo, G. et al., J. Virol., 65:1948-1757 (1991).
The 2-5A system, however, almost certainly provides functions beyond the antipicornavirus activity of interferons. For instance, introduction of 2-5A into cells, Hovanessian, A. G. and Wood, J. N., Virology, 101:81-90 (1980), or expression of 2-5A synthetase cDNA, Rysiecki, G. et al., J. Interferon Res., 9:649-657 (1989), inhibits cell growth rates. Moreover, 2-5A-dependent RNase levels are elevated in growth arrested cells, Jacobsen, H. et al., Proc. Natl. Acad. Sci. USA, 80:4954-4958 (1983b), and 2-5A synthetase, Stark, G. et al., Nature, 278:471-473 (1979), and 2-5A-dependent RNase levels are induced during cell differentiation. See, e.g., Krause, D. et al., Eur. J. Biochem., 146:611-618 (1985). Therefore, interesting correlations exist between 2-5A-dependent RNase and the fundamental control of cell growth and differentiation suggesting that the 2-5A system may function in general RNA metabolism. The ubiquitous presence of the 2-5A system in reptiles, avians and mammalians certainly supports a wider role for the pathway. See, for example, Cayley, P. J. et al., Biochem. Biophys. Res. Commun., 108:1243-1250 (1982).
While it is presently believed that the 2-5A system is the only well-characterized RNA degradation pathway from higher animals, the dsRNA-dependent protein kinase enzyme, known as PKR, is also thought to have antiviral effects in higher animals. Like the 2-5A synthetase enzyme, it is believed that PKR is stimulated by dsRNA. It is believed that activated PKR phosphorylates the alpha subunit of translation factor eIF.sub.2 -alpha, which indirectly inhibits protein synthesis initiation. It is believed that interferons .alpha., and Y induce the accumulation of PKR. See Hovanessian et al.: J. Interferon Res., 9:641-647 (1989).
Like the 2-5A system, the PKR system is also likely to provide functions beyond the antipicornavirus activity of interferons. See Meurs, E. F. et al.: J. Virology, 66:5805-5814 (1992). For example, expression of mutant forms of PKR in NIH 3T3 cells resulted in tumor formation when injected into nude mice. See Meurs, E. F. et al.: Proc. Natl. Acad. Sci USA, 90:232-236 (1993).
In short, the 2-5A system and the PKR system inhibit viral protein synthesis. This is believed to be accomplished by the 2-5A system by degrading mRNA and rRNA whereas the PKR system is believed to accomplish this by indirectly inhibiting protein synthesis initiation.
Viral plant diseases are pandemic and their severity varies from mild symptoms to plant death. The majority of plant viruses are believed to have single stranded RNA genomes. Moreover, it is currently believed that plants are void of the three enzymes discussed above, i.e., PKR, 2-5A synthetase and 2-5A-dependent RNase. See Cayley, P. J. et al.: Biochem. Biophys. Res. Commun., 108:1243-1250 (1982) and Devach, Y. et al.: Biochemistry, 24:593-599 (1985); but see Crum, C. et al.: J. Biol. Chem., 263:13440-13443 (1988); Hiddinga, H. J. et al.: Science, 241:451-453 (1988); Sela, I.: TIBS, pp. 31-33 (February 1981); and Devach, Y. et al.: Science, 216:1415-1416.
The interferon family of cytokines are believed to induce an antiviral state in cells of higher vertebrates. J. Vilcek, G. C. Sen, In Virology, B. Fields, ed. (Raven, New York, 1995). Virus infected cells produce and secrete interferons .alpha. and .beta., which are believed to signal uninfected cells that a virus infection has occurred. Interferons are thought to bind to specific cell surface receptors, activating Jak-STAT signal transduction pathways which induce interferon-stimulated genes. Darnell, J. E. et al.: Science, 264:1415 (1994). The proteins encoded by these genes are believed to be responsible for the biological effects of interferons, including the antiviral responses. The 2-5A system, see Kerr, I. M. et al.: Proc. Natl. Acad. Sci. USA, 75:256 (1978), is one such interferon-induced, antiviral pathway present in cells of reptiles, avians and mammals. Cayley et al.: Biochem. Biophys. Res. Commun., 108:1243 (1982). Two types of enzymes are believed to be essential for a functional 2-5A system: 1) any one of several 2-5A synthetases, which require dsRNA to produce 2-5A, a series of 5'-phosphorylated, 2',5'-linked oligoadenylates, A. G. Hovanessian, A. G. et al.: Nature, 268:537 (1977), and Marie, I. et al.: J. Biol. Chem., 267:9933 (1992); and 2) the 2-5A-dependent Rnase L. Clemens, M. J. et al.: Cell, 13:565 (1978), Slattery et al.: Proc. Natl. Acad. Sci. USA, 76:4778 (1978), and Zhou, A. et al.: Cell, 72:753 (1993). 2-5A is thought to activate Rnase L which cleaves viral and cellular single-stranded RNAs, predominantly after UpNp sequences. Floyd-Smith, G. E. et al.: Science, 212:1020 (1981); and Wreschner, D. H. et al.: Nature, 289:414 (1981). Because virus-infected cells often contain dsRNA activators of 2-5A synthetase, see, e.g., Gribaudo G. et al.: J. Virol., 65:1748 (1991), for instance as replicative intermediates of RNA viruses, RNA degradation by Rnase L is believed to frequently occur in interferon-treated, virus-infected cells. See Wreschner, D. H. et al.: Nucleic Acids Res., 9:1571 (1981); and Silverman, R. H. et al.: Eur. J. Biochem., 124:131 (1982). It is believed that the 2-5A system inhibits replication of the picornaviruses, encephalomyocarditis virus and mengo virus in interferon-treated mammalian cells. Chebath, J. et al.: Nature, 330:587 (1987); Rysiecki, G. et al.: J. Interferon Res., 9:649 (1989); Coccia, E. M. et al., Virology, 179:228 (1990); and Hassel, B. A. et al.: EMBO J., 12:3297 (1993).
RNase L, assayed by 2-5A binding activity, was not detected in tobacco, Nicotiana glutinosa and N. tabacum. See Cayley et al.: Biochem. Biophys. Res. Commun., 108:1243 (1982). Furthermore, 2-5A synthetase was not detected in such plants, and 2-5A synthetase was not detected in control, tobacco mosaic virus (TMV)-infected, interferon-treated or poly (I):poly (C)-treated plants. Plants therefore appear to lack a 2-5A system or an equivalent of the mammalian 2-5A system.
Previously, expression in potato of rat 2-5A synthetase mRNA, without co-expression of RNase L., was correlated with possible partial resistance to potato virus X (PVX) in some, but not all transgenic plants. See Truve, E. et al.: Bio/Technology, 11:1048 (1993). The biochemical basis for both this perceived effect and for a possible anti-TMV effect of 2',5'-oligoadenylate in non-transgenic N. glutinosa leaf disks has not been established. See Y. Devash, et al.: Science 216: 1415-1416 (1982). Although plant oligoadenylates are believed to exist, they appear to differ from 2-5A, see, e.g., Y. Devash et al.: Biochem., 24:593 (1985). It is therefore presently believed that plants do not produce or express 2-5A synthesis and RNase L.
Notwithstanding the importance of 2-5A-dependant RNase to the 2-5A system, 2-5A-dependent RNase enzymes having ribonuclease function have not been isolated, purified or sequenced heretofore. Consequently, there is a demand for isolated, active 2-5A-dependent RNases and their complete amino acid sequences, as well as a demand for encoding sequences for active 2-5A-dependent RNases. There is also a demand for transgenic plants which are immune or resistant to viruses, such as transgenic tobacco plants that are immune or resistant to viruses such as the picornaviruses, tobacco mosaic virus (TMV), tobacco etch virus (TEV) and alfalfa mosaic virus (AIMV).